MOLECULAR ANALYSIS OF ENDOGENOUS CANNABINOID TRANSPORT

描述：(申请人摘要) 此R21应用程序的广泛、长期目标是推动 全面认识内源性大麻素的分子调控机制 通过假定的内源性大麻素摄取系统发出信号。内生性 大麻素是脂肪酸衍生的分子，如花生胺，其 新陈代谢和信号终止似乎依赖于运输回 释放细胞以便随后被细胞内的氨基水解酶分解 酵素。因此，吸收过程将在动态中发挥关键作用。 内源性拟大麻活性的调节。具体目标是(1) 分离鉴定内源吸收相关蛋白(S) 大麻素，以及(2)表征与以下物质有关的运输活动 安非他明摄取。这个项目具有与健康直接相关的内生性 大麻素系统参与调节多种生理和生理功能。 病理过程包括精神病、运动、炎症、血液 压力和痛苦。根据显示功能相似的数据 花青胺转运和长链脂肪酸摄取的研究 设计目的是探索各种脂肪酸的潜在贡献 异源表达在内源性大麻素转运中的作用 系统。此外，内源性大麻素摄取的药理学概况 将确定原生和转基因细胞，并测定其组织分布 对已确认的阿南达胺转运体进行了调查，以更好地了解 转运蛋白在内源性大麻素信号中的作用。将使用的方法 包括分子克隆技术、运输蛋白特性 利用放射性标记底物摄取试验，测定细胞内 内源性大麻碱底物积累的质谱学和原位分析 杂交以揭示转运体的分布。这些研究将提供一个 为进一步研究与此相关的分子机制奠定基础 内源性大麻素转运的结构/功能和调控。

DESCRIPTION: (Applicant's Abstract) The broad, long-term objective of this R21 application is to advance the overall understanding of molecular mechanisms regulating endogenous cannabinoid signaling by a putative endocannabinoid uptake system. The endogenous cannabinoids such as anandamide are fatty acid-derived molecules whose metabolism and signal termination appear to rely upon transport back into releasing cells for subsequent breakdown by an intracellular amidohydrolase enzyme. Thus, the uptake process would play a critical role in the dynamic regulation of endogenous cannabimimetic activity. The Specific Aims are (1) To isolate and identify the protein(s) responsible for the uptake of endogenous cannabinoids, and (2) To characterize the transport activity associated with anandamide uptake. This project has direct health-relatedness as the endogenous cannabinoid system has been implicated in modulating various physiological and pathological processes including psychosis, motor movement, inflammation, blood pressure, and pain. Based upon data indicating functional similarities between anandamide transport and the uptake of long-chain fatty acids, the Research Design is to explore the potential contributions of various fatty acid transporters in endocannabinoid transport using heterologous expression systems. In addition, the pharmacologic profile of endocannabinoid uptake in native and transfected cells will be determined, and the tissue distribution of the identified anandamide transporter investigated to better understand the role of transport proteins in endocannabinoid signaling. The Methods to be used include molecular cloning techniques, characterization of transport proteins using radiolabeled substrate uptake assays, determinations of intracellular accumulation of endocannabinoid substrates using mass spectrometry, and in situ hybridization to reveal transporter distribution. These studies will provide a basis for future studies examining the molecular mechanisms associated with both structure/function and regulation of endogenous cannabinoid transport.